Gen5tm

(±)-Nutlin-3 (Cayman Chemicals, Ann Arbor, MI, USA) was dissolved in DMSO to make 50 mM stock solution. MI-773 (MedChem Express Co. Ltd., China) and AMG232 (MedChem Express) were dissolved in DMSO to make 10 mM stock solutions. Overnight yeast cultures were washed and then diluted to OD₆₀₀ = 0.2 in 1 mL SD-Leu-Trp-His-Ade medium containing the desired concentration of the drug. The cultures were then placed in a 30 °C incubator with shaking for 3 days. Growth of the cultures was monitored by measuring the absorbance at 600 nm using the Implen Nanophotometer. This assay was also performed in a 96-well plate format with 200 μL SD-Leu-Trp-His-Ade medium containing the desired concentration of the drug in duplicate. OD₆₀₀ at different time points was measured using a Gen 5^TM (BIO-TEK Instrument, Vermont, USA) microplate reader. The average of the two OD₆₀₀ readings was calculated and used in the analysis.

TPC was determined by the Folin–Ciocalteu method in 96-well plates by using a BioTek Synergy HT multi-mode microplate reader with BioTek’s Gen5TM software (BioTek Instruments Inc., Winooski, VT, USA). Data were expressed as gallic acid equivalents. The antioxidant and antiradical power of the grape samples as well as the digestion products in the different phases were determined by two methods, the DPPH method and the FRAP method. Both methods were executed in the same way as the previous method, in 96-well plates and using the same microplate reader as before [8 (link)]. All analyses were performed in triplicate and in every case, average and standard deviation values were calculated.

BY4743 cells from an overnight culture were diluted to OD_{600 nm} of 0.0625 in YPD-HEPES medium. 200 μL of the diluted yeast culture was transferred into the 96-well microtiter plate having various concentrations of auroramycin and hygromycin. Cells were incubated in a microplate reader for 16–24 hours at 30 °C with shaking. Growth of the cultures was recorded by measuring the OD_{600 nm} using the microplate reader Gen 5^TM (BIO-TEK Instrument, Vermont, USA). The bliss model assumes that the responses are independent events, such as distinct mode-of-actions, and uses a probabilistic calculation (E_A+E_B(1-E_A)) to an expected combined effect of drug A(E_A) and drug B (E_B). Synergy is given by the percent excess of the bliss calculation. Heatmaps were generated using Shinyheatmap [41 (link)].

The method described by Dávalos et al. was employed [48 (link)]. A hydroxyl radical (OH^●) is generated in a living organism, having important negative effects in inflammatory processes of tissue illnesses related to oxidative stress.
For ABTS and ORAC analyses, a multimodal plate reader SynergyTM HT with an automatic dispenser of samples, and temperature control from Biotek Instruments (Winooski, VT, USA) was used. The software Biotek Gen5TM was used for data analysis. Each plate with 96 wells was analyzed in quadruplicate, with four standard levels of calibration and 8 repetitions for blank or control. The reaction was started by the automatic addition of 60 μL of ABTS radical or AAPH to the sample solution for ABTS and ORAC assays, respectively. Antiradical activity with ABTS was determined after 10 min of reaction. For ORAC method, the value was read after 180 min of reaction. ABTS activity was determined in quadruplicate and ORAC activity in triplicate.

The 2,2-diphenyl-1-picrylhydrazyl (DPPH^•) scavenging assay was performed as reported before [57 (link)], with some modifications. A volume of 25 μL of extract serial dilutions was mixed with 200 μL of 100 μM DPPH^• reagent, freshly prepared in methanol [58 (link)] in a 96-well plate. GA was used as positive control, and DMSO as negative control. The plate was incubated in the dark, at room temperature, for 15 min. The absorbance of the extracts (dilutions prepared in DMSO) was measured at 515 nm using a Synergy HT Multi-detection Microplate Reader operated with GEN5^TM (Biotek, Bad Friedrichshall, Germany). Three independent assays were performed in duplicate. Results were expressed as percentage of radical scavenging face to the untreated control.

The TPC of the cyanobacterial extracts was determined using the colorimetric assay of Folin-Ciocalteu, according to Barroso et al. [56 (link)], with some modifications. Briefly, a volume of 25 μL of each extract (10 mg mL⁻¹) was thoroughly mixed with 25 μL of Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA), 200 μL of Na₂CO₃ solution (75 g L⁻¹) and 500 μL of deionized water. After the incubation period (60 min at room temperature), the absorbance of the colored product was measured at 725 nm, using a Synergy HT Multi-detection microplate reader operated by GEN5^TM (Biotek, Bad Friedrichshall, Germany). A standard calibration curve (y = 1.951x + 0.01135; R² = 0.9989) was obtained with seven concentrations of gallic acid (GA) (0.025 to 0.5mg mL⁻¹). Total phenols in each extract were expressed in mg gallic acid equivalents (GAE) g⁻¹ dry biomass. The experiment was carried out in triplicate.